Biofilm reduction in crossflow filtration systems

ABSTRACT

Systems and methods for reducing biofilms are described. The systems and methods are particularly suitable for use with conventional aqueous crossflow filtration systems, such as reverse osmosis systems. The addition of the enzyme/surfactant compound has been found to enhance the effectiveness of conventional crossflow filtration processes by decreasing or inhibiting the growth of biofilms and other contaminants.

FIELD OF THE INVENTION

The present invention relates to systems and methods for reduction andcontrol of bacterial biofilm. More particularly, the present inventionrelates to systems and methods for reduction of biofilms in crossflowfiltration systems.

BACKGROUND OF THE INVENTION

Biofilms are formed when colonies of bacteria aggregate on surfaces inmany different locations. When bacteria in biofilm aggregate, theyproduce a sugary, polysaccharide-containing mucous coating, or slime.Bacteria grow and multiply faster when attached (sessile) than whenfree-floating (planktonic). Within the slime, the bacteria form complexcommunities with intricate architecture including columns, waterchannels, and mushroomlike towers. These structural details are believedto improve biofilm nutrient uptake and waste elimination, as bloodvessels do in an animal's body. More information about biofilms isprovided in an article entitled “Sticky Situations: Scientists areBeginning to Understand How Bacteria Find Strength in Numbers” by JessaNetting, published in Science News, 60:28-30, Jul. 14, 2001, which isthe basis for the information in this and the following paragraphs, andwhich is hereby incorporated by reference herein in its entirety.

Biofilms occur in a wide range of locations. Many are found on or in thehuman body, including on the teeth, gums, ears, prostate, lungs, andheart, where they are believed to be implicated in chronic infectionssuch as gum disease, ear infections, infections of the prostate glandand heart, and lung infections in people with cystic fibrosis. Biofilmsalso occur in nature, such as the slime that covers river rocks,marshes, and the like. Biofilms also occur in medical equipment, such ascatheters, and are a major source of hospital infections. Biofilms canalso occur in areas such as contact lenses, other medical equipment, andin other industries. A primary difficulty with biofilms is that they aremore difficult to reduce or eliminate than are individual bacteria. Thisis due to the formation of the protective layer of slime, as well asadaptations that the individual bacteria undergo when they formbiofilms.

One important area in which biofilms occur is in aqueous systems thatuse separation membranes, such as particle filtration, microfiltration,ultrafiltration, nanofiltration, and particularly reverse osmosis (“RO”)systems. Microfiltration membranes are typically polymer or metalmembrane disc or pleated cartridge filters rated in the 0.1 to 2 micronrange that operate in the 1 to 25 psig pressure range. Ultrafiltrationis a crossflow process that rejects contaminants (including organics,bacteria, and pyrogens) in the 10 angstrom to 0.1 micron range usingoperating pressure in the 10 to 100 psig range. Nanofiltration equipmentremoves organic compounds in the 200 to 1,000 molecular weight range,rejecting selected salts. Reverse osmosis removes virtually all organiccompounds and 90 to 99% of all ions under pressure in the 200 to 1000psig range.

These systems use membranes to selectively remove or separate extremelysmall substances from water and process streams in residential,commercial, and industrial applications. When biofilm is present on themembrane due to microbial growth, colloidal solids and insolubleprecipitates can adhere to the sticky substance. As this combinationbuilds, water transmission rates through the membrane are reduced and/oradditional pressure must be applied to maintain the same watertransmission rates. Colloidal solids, microbiological growth, andinsoluble precipitates can collect on the membrane during operation.Conventional treatment methods include continuous dosing, in which aresidual level of a biocidal agent is maintained within the system, orperiodic cleaning and sanitization, in which the filtration system isshut down for a periodic cleaning and sanitization using biocidalagents, acids and caustics. Even with continuous dosing methods, at somepoint the filtration system must be shut down so that the membrane canbe cleaned or replaced. This results in downtime and consequentadditional operating expense. Moreover, the cleaning and biocidal agentsand caustics that are conventionally used to clean and sanitize thefiltration systems have the effect of degrading the filter membranes,which are typically comprised of polymers such as cellulose acetate orpolyamide polymers. A number of pre-treatment processes are alsoavailable to reduce the fouling potential of the feed water beingintroduced to the membrane. These include various types of filtration,disinfection, and chemical treatment. Even with these methods, however,most RO treatment systems must be cleaned regularly.

Accordingly, there exists a long-felt need for improved treatmentprocesses that can achieve reductions in biofilm, particularly in thearea of aqueous systems that use separation membranes.

SUMMARY OF THE INVENTION

The present systems and methods are directed to the use of compositionsincluding enzymes and surfactants to obtain biofilm reduction. Theenzyme/surfactant compound comprises a blend of enzymes and surfactantsover a broad range of compositions and concentrations dependent upon thebiofilm treatment application. Additional optional components mayinclude micronutrients that are generated during the enzyme productionprocess or added as additional ingredients. Further optional ingredientsin the enzyme/surfactant compound include enzyme stabilizers andanti-microbials to prevent product degradation.

The addition of the enzyme/surfactant compound has been found to enhancethe effectiveness of crossflow filtration systems, particularly reverseosmosis systems, by increasing throughput, maintaining or improvingefficiency, reducing biofilm, decreasing periodic maintenancerequirements, and decreasing the need for costly system shutdowns.

It is thus an object of this invention to provide systems and methodsfor reducing biofilms.

It is a further object of this invention to provide systems and methodsfor improving the performance of aqueous systems that use separationmembranes, thus enhancing the separation process of such systems.

It is a still further object of this invention to provide systems andmethods for reducing biofilm and other fouling agents in crossflowfiltration systems, thus enhancing the filtration process.

It is a still further object of this invention to provide improvedaqueous filtration systems and methods that reduce fouling.

These and further objects and advantages will become apparent uponconsideration of the detailed description and drawings enclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram illustrating a conventional reverse osmosisfiltration system.

FIG. 2 is a graphical illustration of a membrane showing its function offiltering an aqueous stream containing contaminants.

FIG. 3 is a graph showing flux vs. time for an aqueous crossflowfiltration system.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The materials, systems, and methods of the present invention involve theuse of an enzyme/surfactant blend compound to reduce biofilms where theyoccur. The description below will focus on crossflow filtration systems,particularly reverse osmosis systems. It should be understood, however,that the materials, systems, and methods described herein have a broadrange of applications, and are not limited solely to filtration systems,crossflow filtration systems, or reverse osmosis filtration systems.

FIG. 1 is a flow diagram illustrating a conventional reverse osmosisfiltration system. Feed water (F) enters a first end of the vessel 10,and concentrate (C) exits from the opposite end of the vessel. Amembrane system 12 will typically comprise a plurality of spiral-woundmembranes tightly wound within the vessel. A permeate tube 14 isprovided at the center of the vessel, surrounded by the membrane system12. As the feed water (F) enters the vessel 10, it encounters themembrane system 12. The permeate (P) passes through the membranes intothe permeate tube 14, where it eventually exits the vessel as indicatedin FIG. 1.

FIG. 2 is a graphical illustration of a membrane showing its function offiltering an aqueous stream. The membrane includes a solid barrier 16,usually formed of cellulose acetate, metal, or a polymer such as apolyamide. Sub-microscopic pores 18 are sized to pass water 20 whilerejecting oil, dirt, and other contaminants 22. The pores also rejectbacteria 24. A properly designed membrane and system allows only desiredmolecules to pass through the membrane barrier regardless of the feedstream contaminant level.

As biofilm or other contaminants build up on a membrane system, thesystem performance will deteriorate. For example, the filtration systemmay require an increased pressure differential to produce the same fluxas the system in its “clean” state. Stated otherwise, for the same levelof pressure differential, the flux rate of the system will decrease. Forthese purposes, the term “pressure differential” or “Delta Pressure”refers to the difference in pressure between the feed stream (F) and thepermeate stream (P), and “flux” refers to the flow rate of the permeatestream (P). This deterioration of performance is illustrated graphicallyin FIG. 3. For each cycle between periodic cleanings, the flux willgradually deteriorate over time as biofilm and other contaminants buildup on the membrane system. The periodic cleaning will cause the fluxlevel to increase, although typically not to its peak level from theprevious cycle because the membrane will generally degrade due to useand, in most circumstances, due to the cleaning process. Thus, inaddition to the fluctuations of the system flux between periodiccleanings, there will also be an observed general decline in systemperformance over time, as illustrated in the parallel downward slopingphantom lines in FIG. 3.

It has been found that the addition of an enzyme/surfactant compound tothe feed stream (F) has the effect of decreasing fouling of the filtersystem, and enhancing the filtration and concentration processes ofconventional filtration systems. The enzyme/surfactant compound is ablend of enzymes and surfactants of a range of compositions andconcentrations. The basic enzyme/surfactant composition may besupplemented with micronutrients generated during the enzyme productionprocess or added separately. It is also possible to supplement theenzyme/surfactant compound with stabilizers, compounds that givesignificant stability to the activity of the enzymes. The compositionmay also optionally be supplemented with anti-microbial agents thatinhibit the growth of microbes in its concentrated form.

Enzymes degrade pollutants of biological origin, such as fats, oils,proteins and polysaccharides. Other enzymes are known to degradehydrocarbons. Enzymes pre-digest pollutants so they may be more easilytaken up and degraded by bacteria. In its preferred form, theenzyme/surfactant compound may contain one purified enzyme or a broadspectrum of enzymes and enzymatic activities. Typical enzymes includedin the compound include lipases and esterases, phosphatases, proteases,glycosidases, cellulases, cellobiases, and polysaccharide hydrolases.Enzymes in the compound may also include enzymes with otherspecificities such as oxidative enzymes, and the composition of anenzyme cocktail used in the compound may be specifically formulated tomeet the needs of specific aqueous filtration applications. Anonlimiting list of enzymes and enzyme activities believed to be usefulin the enzyme/surfactant compound includes the following: alkalinephosphatase, esterase (C-4), esterase-lipase (C-8), lipase (C-14),leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin,chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, alphagalactosidase, beta galactosidase, beta glucuronidase, alphaglucosidase, N-acetyl-beta-glucosaminidase, alpha mannosidase, and alphafucosidase.

There are a great many materials derived from plant, animal, andmicrobial sources that have been known to those skilled in the art to berich sources of enzymes. These source materials may be used with full,partial, or no purification of enzymes to obtain enzymes for use in theenzyme/surfactant compound. Some of these sources of enzymes areprovided in Table 1 below. Additional information concerning the sourcesof enzymes useful in the enzyme/surfactant compound may be found in:Thomas E. Barman, Enzyme Handbook, Vols. I-II (Springer-Verlag, New York1969); Dixon and Webb, Enzymes, pp. 671-785 (Academic Press Inc., 1964);Kornberg, For the Love of Enzymes: The Odyssey of a Biochemist, p. 37(Harvard University Press, 1989); Fruton and Simmonds, GeneralBiochemistry, pp. 218-219 (John Wiley and Sons, 2nd Ed. 1958); U.S. Pat.No. 4,891,320 to Aust et al.; and U.S. Pat. No. 3,635,797 to Battistoniet al. Each of these publications is hereby incorporated by referenceherein in its entirety.

TABLE 1 SOURCE MATERIAL ENZYMES REFERENCE Yeast Extract Invertase(Sucrase) Kornberg Catalase Fruton and Simmonds Lactase Battistoni etal. Maltase Carboxylase Oxidative and Metabolic enzymes Malt ExtractAmylase Kornberg Maltase Battistoni et al. Diastase Hen's Egg WhiteLysozyme Fruton and Simmonds Animal Pancreas Chymotrypsin Fruton andSimmonds Trypsin Plant Juices and Papain Fruton and Simmonds ResinsPeroxidases Bacteria Glycoside Hydrolases Dixon and Webb Proteases andPeptidases Lipases Fungi Peroxidases Aust et al.

The extraction of enzymes from source materials may be enhanced byenzymatic activity. It is a well established practice to effect therelease of enzymes from yeast cells by autolysis, that is the use ofendogenous (yeast produced) enzymes to break yeast cell membranes. Therelease of enzymes may also be enhanced by the use of exogenous enzymes.For example, the addition of cellulases to malt increases the yield ofother hydrolytic enzymes derived from this source material. Beyond this,methods of obtaining or manufacturing enzymes or enzyme cocktails fromthese source materials are well-known in the art and are beyond thescope of this discussion, and will therefore not be repeated here. Allof the enzymes, endogenous, exogenous, and those released from sourcematerials may be incorporated into the composition so that it has asbroad a range of enzymatic activities as possible.

In a preferred form, the enzyme/surfactant compound includes enzymeactivities from Esterases, such as esterase (C-4) and esterase-lipase(C-8); Proteases, such as cystine arylamidase and chymotrypsin;Glycosidases, such as beta galactosidase, beta glucoronidase, betaglucosidase, and alpha mannosidase; and Phosphatases, such as acidphosphatase and naphthol-AS-BI-phosphohydrolase. It is believed thatother and further enzymes may further enhance the effectiveness of theenzyme/surfactant compound. Accordingly, this list should be consideredillustrative and not limiting.

Enzymes may be prepared by a number of methods known to those skilled inthe art, including various fermentation processes. Enzymes for use inthe enzyme/surfactant compound may be prepared as an enzyme cocktailderived from the fermentation product of molasses and diastatic malt bySaccharomyces cerevisiae. Additional yeast strains that may be usedinstead of or in addition to Saccharomyces cerevisiae includeKluyveromyces marxianus, Kluyveromyces lactis, Candida utilis (Torulayeast), Zygosaccharomyces, Pichia, Hansanula, and others known to thoseof skill in the art. Micronutrients may be added to the process,including diammonium phosphate, ammonium sulfate, magnesium sulfate,zinc sulfate and calcium chloride. Additional micronutrients, such asvitamins and amino acids, are produced during the fermentation.

Surfactants that are useful in the enzyme/surfactant compound may beeither nonionic, anionic, amphoteric or cationic, or a combination ofany of the above, depending on the aqueous filtration application.Suitable nonionic surfactants include alkanolamides, amine oxides, blockpolymers, ethoxylated primary and secondary alcohols, ethoxylatedalkylphenols, ethoxylated fatty esters, sorbitan derivatives, glycerolesters, propoxylated and ethoxylated fatty acids, alcohols, and alkylphenols, glycol esters, polymeric polysaccharides, sulfates andsulfonates of ethoxylated alkylphenols, and polymeric surfactants.Suitable anionic surfactants include ethoxylated amines and/or amides,sulfosuccinates and derivatives, sulfates of ethoxylated alcohols,sulfates of alcohols, sulfonates and sulfonic acid derivatives,phosphate esters, and polymeric surfactants. Suitable amphotericsurfactants include betaine derivatives. Suitable cationic surfactantsinclude amine surfactants. Those skilled in the art will recognize thatother and further surfactants are potentially useful in theenzyme/surfactant compound depending on the particular aqueousfiltration application.

Preferred anionic surfactants used in the enzyme/surfactant compoundinclude CalFoam ES 603, a sodium alcohol ether sulfate surfactantmanufactured by Pilot Chemicals Co., and Steol CS 460, a sodium salt ofan alkyl ether sulfate manufactured by Stepan Company. Preferrednonionic surfactants used in the enzyme/surfactant compound includeNeodol7 25-7 or Neodol7 25-9, which are C₁₂-C₁₅ linear primary alcoholethoxylates manufactured by Shell Chemical Co., and Genapol7 26 L-60,which is a C₁₂-C₁₆ natural linear alcohol ethoxylated to 60E C cloudpoint (approx. 7.3 mol), manufactured by Hoechst Celanese Corp. Itshould be understood that these surfactants and the surfactant classesdescribed above are identified only as preferred materials and that thislist is neither exclusive nor limiting of the enzyme/surfactantcompound.

One or more additional components may optionally be added to theenzyme/surfactant compound in order to stabilize the compound andincrease shelf life. Such additional components may include enzymestabilizers, anti-microbial agents, and antioxidants.

Enzyme stabilizers are effective to extend the enzymatic activity ofenzymes. Enzyme stabilizers can include sugars, polyhydrilic alcohols,other organic solvents, ionic or nonionic species, and polymers. Anexample of a commonly used stabilizer is propylene glycol.

Examples of anti-microbial agents that may be used in theenzyme/surfactant compound include propylene glycol, methyl paraben,propyl paraben, and sodium benzoate.

Examples of antioxidants that may be incorporated into theenzyme/surfactant compound include butylated hydroxyanisole (BHA),butylated hydroxytoluene (BHT), ascorbic acid, and others.

It is preferable to adjust the pH of the enzyme/surfactant compound tofrom about 3.75 to about 5.0, and most preferably to from about 4.2 toabout 4.5, with phosphoric acid to help stabilize the product. Inparticular, it is beneficial to make the compound acidic in order tooptimize the activity of the anti-microbial agents, such as methyl orpropyl paraben.

A preferred composition of an enzyme/surfactant compound useful in thesystems and methods described herein is provided in Table 2 below.

TABLE 2 Concentra- Preferred tion Range Concentra- (% by tion (%Component Type Component weight) by weight) Solvent Water 25.0-90.063.92 Enzyme/Nutrient Enzyme Cocktail  5.0-60.0 20.0 Source(Fermentation product of molasses and diastatic malt by Saccharomycescerevisiae) Micronutrients Inorganic salts 0.05-2.50 0.31 (e.g.,diammonium phosphate, ammonium sulfate, magnesium sulfate, zinc sulfate,calcium chloride) Surfactant Neodol7 25-7  2.0-40.0 7.5 (Non-ionic)Surfactant (Anionic) Steol CS 460  0.5-20.0 2.5 Stabilizer Propyleneglycol  0.5-40.0 5.27 Anti-microbial agent Methyl paraben 0.03-0.5  0.15Anti-microbial agent Propyl paraben 0.01-0.3  0.05 Anti-microbial agentSodium benzoate 0.03-0.5  0.15 Antioxidant BHA 0.002-0.1  0.02Antioxidant BHT 0.002-0.1  0.02 Antioxidant Ascorbic acid 0.05-2.0  0.11TOTAL: 100.00

To achieve reduction of biofilm and other contaminants in a reverseosmosis or other aqueous crossflow filtration system, anenzyme/surfactant compound, such as one having the composition listed inTable 2 above, is preferably added to a feed stream at a concentrationof about 0.1 parts per million (ppm) to about 25 ppm, depending on theparticular application and the contaminants of interest. Higher or lowerconcentrations may also be possible. A preferred concentration fortertiary treatment of municipal wastewater is from about 0.5 ppm toabout 5 ppm, particularly about 3 ppm. A cleaning cycle may be employedwherein the concentration is increased for a period of time. A preferredcleaning cycle includes a concentration of the enzyme/surfactantcompound of about 6 to about 25 ppm for a period of from about 3 toabout 12 hours. A particularly preferred cycle comprises a concentrationof about 9 ppm for a period of about 6 hours.

Experiments have shown that the use of the enzyme/surfactant compoundincreases system performance by preventing fouling due to biofilm andother contaminants. This result has been observed in two ways. Referringonce again to FIG. 3, first, the flux rate between system cleaningcycles has been observed to decrease less through use of theenzyme/surfactant compound. Second, the overall system performance hasbeen observed to degrade significantly less when the enzyme/surfactantcompound has been used. It is believed that the increased systemperformance may be due, at least in part, to a breakdown of the stickypolysaccharide biofilm material.

For example, studies were conducted to determine the effects of treatingsludge with the composition listed as the “Preferred Concentration” inTable 2. Sludge mass is made up of clumps of bacteria, held together bythe sticky polysaccharide biofilm material. During the filtrationprocess, this creates a barrier film that does not allow the water tofreely drain from the sludge mass. Sludge was obtained from the OrangeCounty (California, United States) Sanitation District for the study.Testing was conducted to determine TSS (Total Suspended Solids), COD(Chemical Oxygen Demand), and Filtration Characteristics, or waterretention. Baseline data, as well as treated and control were recorded,with the following results:

Sludge Volume TSS (%) COD (ppm) Reduction (%) Baseline 3.82%  390 ppmN.A. Control 3.59% 2115 ppm 20.33% Treated 3.59% 2590 ppm 14.53% %Change Over 0.00% +18.3% −28.5% Control

While the TSS levels were reduced by 7% for both the Treated and Controlfrom the Baseline, the permeate from the Treated sample exhibited anincrease in COD of 18.3% over the Control. It is assumed that thereduction in TSS occurred due to a decay of the actual cells or walls.However, the COD results, in conjunction with the significant reductionof sludge volume for the Treated sample vis-à-vis the Control, establishthat channeling in the sludge matrix is improved, allowing the water todrain through (or around) the bacterial cell matter. This is due to thebreakdown of the sticky polysaccharide biofilm material.

The foregoing descriptions include a detailed description of the use ofan enzyme/surfactant compound for the treatment of wastewater ordrinking water in crossflow filtration systems. One example of suchsystems is water desalination. It has been found that theenzyme/surfactant compound, when used in a water desalination process,improves the efficiency of the desalination process.

In the wastewater or drinking water systems and methods describedherein, the filtrate is the “product” obtained from the permeate in theprocess. As yet another example, the methods described herein may alsobe used in applications where the concentrate is the “product.” Forexample, in the dairy, fruit juice, or other industries, concentrationprocesses are used to eliminate water from a feed stream. In those typesof systems and methods, the enzyme/surfactant compound may be used aspart of a cleaning cycle to effectively clean filter membranes withoutdamaging them as would be the case with strong acid or caustic cleaningmaterials. In those systems and methods, the enzyme/surfactant compoundis circulated/recirculated at a concentration of as low as 5-10 ppm toas high as 1% to 5%. The actual use levels would be highly dependent onthe nature of the foulant, time limits for the cleaning cycle,temperatures of the cleaning solutions, filter membrane constructionand/or materials, filter type (micro, ultra, nano, or reverse osmosis),and other factors. As a non-limiting example, the process ofconcentrating milk causes a significant buildup of not only biofilm onthe filter membranes but also significant quantities of butter fat, oil,protein, and other milk constituents. In that example, a circulation ofa cleaning solution of about 1% to 2% of the enzyme/surfactant compoundis utilized at a temperature of 100° F. or higher for as little as 10minutes. Treatments using the enzyme/surfactant compound result inreduced wear and tear on the filter membranes.

Yet another application of the enzyme/surfactant compound is inconnection with filtration membranes used to eliminate the use ofsecondary clarifiers in wastewater treatment systems. In such systems,the enzyme/surfactant compound is introduced in, or prior to, theaeration basin. The filtration membranes are also placed in the aerationbasin, but are placed in a quiescent zone within the basin. The benefitsof using the enzyme/surfactant compound in these systems are: 1) toincrease metabolic activity during the aerobic process, 2) to increaseoxygen uptake, thus reducing aeration power requirements and costs, 3)to reduce biomass production, and 4) to reduce or eliminate biofoulingof the filtration membranes.

Accordingly, the systems and methods described herein achieve improvedreduction of biofilms and other benefits. While the above descriptionhas focused primarily on crossflow filtration systems, and particularlyreverse osmosis systems, it is believed that the enzyme/surfactantcompound is useful in reducing biofilms wherever they may occur.

Thus, the compounds, systems and methods of the present inventionprovide many benefits over the prior art. While the above descriptioncontains many specificities, these should not be construed aslimitations on the scope of the invention, but rather as anexemplification of the preferred embodiments thereof. Many othervariations are possible.

Accordingly, the scope of the present invention should be determined notby the embodiments illustrated above, but by the appended claims andtheir legal equivalents.

What is claimed is:
 1. A method for reducing biofilm in an aqueoussystem, comprising the steps of: providing a mixture containing enzymesand surfactants, wherein said mixture comprises lipase, and introducingthe mixture to an aqueous system containing biofilm.
 2. The method ofclaim 1, wherein said mixture comprises at least one nonionicsurfactant.
 3. The method of claim 2, wherein said mixture comprises atleast one nonionic ethoxylated primary alcohol.
 4. The method of claim1, wherein said aqueous system is a crossflow filtration system.
 5. Themethod of claim 4, wherein said mixture comprises a fermentation productSaccharomyces cerevisiae.
 6. The method of claim 5, wherein said mixturecomprises at least one nonionic surfactant.
 7. The method of claim 5,wherein said mixture further comprises a C₁₂-C₁₆ linear alcoholethoxylate surfactant.
 8. The method of claim 7, wherein saidfermentation product is present in said mixture at a concentration offrom about 5.0% by weight to about 60.0% by weight, and said mixture isadded to the reverse osmosis system to obtain a concentration by weightof the mixture of from about 0.1 part per million to about 25 parts permillion.
 9. The method of claim 7, wherein said fermentation product ispresent in said mixture at a concentration of from about 5.0% by weightto about 60.0% by weight, and said mixture is added to the reverseosmosis system to obtain a concentration by weight of the mixture offrom about 1 parts per million to about 5 parts per million.
 10. Amethod for reducing biofilm in an aqueous system, comprising the stepsof: providing a mixture containing enzymes and surfactants, wherein saidmixture comprises a fermentation product of Saccharomyces cerevisiae,and introducing the mixture to an aqueous system containing biofilm. 11.The method of claim 10, wherein said mixture comprises at least onenonionic ethoxylated primary alcohol surfactant.
 12. The method of claim10, wherein said mixture comprises a C₁₂-C₁₆ linear alcohol ethoxylatesurfactant.
 13. The method of claim 12, wherein said fermentationproduct is present in said mixture at a concentration of from about 5.0%by weight to about 60.0% by weight, and said mixture is added to theaqueous system to obtain a concentration by weight of the mixture offrom about 0.1 part per million to about 25 parts per million.
 14. Themethod of claim 12, wherein said fermentation product is present in saidmixture at a concentration of from about 5.0% by weight to about 50.0%by weight, and said mixture is added to the aqueous system to obtain aconcentration by weight of the mixture of from about 1 parts per millionto about 5 parts per million.
 15. The method of claim 12, wherein saidfermentation product is present in said mixture at a concentration offrom about 5.0% by weight to about 60.0% by weight, and said mixture isadded to the aqueous system to obtain a concentration by weight of themixture of from about 1% to about 2%.
 16. The method of claim 15 whereinthe system temperature is 100° F. or higher.
 17. The method of claim 16comprising the additional step of removing the mixture containingenzymes and surfactants from the aqueous system within 10 minutes ofsaid introducing step.